X-ray crystallography is an established, well-studied technique for providing a three-dimensional representation of the appearance of a molecule in a crystal. This technique remains one of the most powerful tools for atomic resolution determination of molecular structures. The crystalline atoms in a structure cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information.
Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic and biological molecules—scientists have employed X-ray crystallography to determine the crystal structures of many molecules. The method reveals the structure and function of many biological molecules, including vitamins, drugs, small molecules, ligands, proteins, polypeptides, and nucleic acids. X-ray crystal structures can also account for unusual electronic or elastic properties of a material, shed light on chemical interactions and processes, or serve as the basis for designing pharmaceuticals against diseases.
In order to perform an X-ray crystallographic analysis, a beam of X-rays strikes a single crystal, producing scattered beams. When they land on a piece of film or other detector, these beams make a diffraction pattern of spots; the strengths and angles of these beams are recorded as the crystal is gradually rotated. Each spot is called a reflection, since it corresponds to the reflection of the X-rays from one set of evenly spaced planes within the crystal. The atoms in a crystal are not static, but oscillate about their mean positions, usually by less than a few tenths of an angstrom. X-ray crystallography allows measuring the size of these oscillations.
X-rays that are diffracted from a crystal of a molecule give rise to a pattern of diffraction “spots”, with each spot corresponding to a point in the reciprocal crystal lattice, representing a wave with an amplitude and a relative phase. Structure factors corresponding to the reciprocal crystal lattice also correspond to the electron density distribution within the crystallographic “unit cell.” Electron density corresponding to the structure factors can be determined by an inverse Fourier transformation. The calculation of a useful electron density map requires combining the observed amplitudes with correct phases.
Determination of the phase for structure factors remains the most challenging obstacle for the crystallographic analyses of molecules, such as proteins, and complexes. New methods to enable the determination of phases for X-ray diffraction data to be used in structural analyses are sought.
Nucleic acids play a variety of important roles in biological systems, including the transfer and regulation of genetic information (Ban et al., Science, 289:905-920 (2000)). Moreover, nucleic acids, especially RNAs, can fold into well-defined three-dimensional structures and catalyze biochemical reactions in processes such as protein synthesis and in the life cycle of some viruses. RNAs and DNAs with catalytic and binding functions have also been identified via in vitro selection. Furthermore, the recent discovery of noncoding small RNAs in diverse organisms has enormously expanded the repertoire of functions of nucleic acids (Storz, Science, 296:1260-1263 (2002); Lee et al., Science, 294:862-864 (2001)). This vast array of biologically active RNAs and DNAs has promoted a new front of research in the field of structural analysis to elucidate their three-dimensional structure and functional relationships.
As ubiquitous biological molecules in all living systems, nucleic acids are important drug targets, and they can also be used in diagnostics and therapeutics.
The X-ray crystallographic analyses of nucleic acids and the molecules to which they bind are often difficult and can be time and labor-intensive, owing to problems with the production and diffraction quality of crystals containing molecule/nucleic acid complexes.
Therefore, it is an object of the invention to provide compositions and methods for the production of crystals and determination of X-ray crystal structures of molecules of interest, such as proteins, complexed with nucleic acids.
It is another object of the invention to provide compositions and methods for selecting and designing molecules that can bind to molecules of interest, such as proteins, that are or can complex with nucleic acids.
It is another object of the invention to provide complexes of molecules of interest, such as proteins, and selenium-derivatized nucleic acids.